Slide 1

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 2

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 3

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 4

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 5

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 6

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 7

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 8

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 9

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 10

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 11

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 12

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 13

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 14

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 15

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 16

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 17

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 18

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 19

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 20

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 21

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 22

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 23

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 24

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 25

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 26

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 27

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 28

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 29

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 30

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 31

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 32

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 33

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 34

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 35

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 36

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 37

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 38

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 39

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 40

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 41

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 42

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.


Slide 43

Searching for microbes
Part XIV.

The Biofilm
Ondřej Zahradníček
To practical of VLLM0421c
[email protected]

Survey of topics
Clinical cases related to biofilm
Characterisation of biofilm

Diagnostic and experimental method for biofilm
Pictures of biofilm

Clinical cases
related to biofilm

Story one (today a real one)
• Male, 58 let, 2001 cardiostimulator, 2002
repeatedly hospitalized on an internal
department with fever of unknown
origin, elavation of inflamatory markers
• In blood cultures, S. epidermidis, very
good susceptibility
• Several times treated by high doses of
antibiotics in combinations (oxacilin,
gentamicin, rifampicin, cefazolin,
cefalotin, clindamycin)

Story – contiuing
• In the beginning, a good response, later attacks of
fever again
• At transoesofageal examination, vegetation on a
chamber electrode sized 1,5 × 1,5 cm.
• Cardiologists repeatedly refuse cardiostimulator
removal. A combination oxacilin + gentamicine +
rifampicine, pacient in a good state.
• Nevertheless, again temperature and CRP rises.
Vancomycin and rifampicin starts to be used, after
improval, patient‘s trombus is removed and the
electrode changed (under antibiotics), so the
patient starts to be better.

Who is guilty? The biofilm!!!






The therapy could not be successfull, because
high resistance of bacteria growing in form
of a biofilm was not taken into account.
The therapy was not strong enough from the
beginning and the biofilm was not
erradicated.
Only electrod removal (under antibiotic
therapy) enabled pacient status improval.

Catheter biofilm

webs.wichita.edu

Story two
• Michal was a 13-year old boy. He hated his parents
and he decided to do anything against them.
• He decided that one of methods to fight them could
be to resist everything his parents insist on him to do.
• So he decided to stop cleaning his teeth, keeping
order in his room and some more activities like that.
• But very soon, a toothache started. He had to visit a
dentist. The dentist said he has a severe dental
caries. She repaired his teeth, but also wanted him to
clean his teath again, otherwise he would have
problems again – not only with her (and other „comeback-to-your-grave generation), but mostly with his
own teeth.

The criminal agent was
• …an overgrown biofilm again.
• In oral cavity, a biofilm is normal. It is
even useful: the normal oral microflora is
organised in it, and so it is more resistant to
outer influences, including pathogenic agents
• Nevertheless, too overgrown biofilm (as a
result of too many sugars eaten and too little
teeth cleaning) makes oral biofilm to be an
enemy instead to be a friend of a patient.

Biofilm missed by a toothbrash
may lead to a caries formation

3× webs.wichita.edu

Characterisation of
biofilm

Biofilm: what is it?
• A biofilm is a complex, organized
structure
• It consists of living cells (mostly bacteria),
masses produced by them (mostly
polysaccarides) and channels
• It is present not only inside living body, but
also in the environment. For example
stones in ponds and rivers are often covered
by a biofilm that makes them smooth.

Biofilm in a river

www.sbs.soton.ac.uk

Various pictures of biofilm
Biofilm on a cathetre

Photo: Archive of Veronika Holá

Various pictures of biofilm
Biofilm on a catheter
Bacteria
A channell
Catheter
Polysaccharides

Photo: Archive of Veronika Holá

biology.fullerton.edu

Stages of biofilm development
• Direct contact of a planctonic bacteria
with a surface
+
• Adhesion to this surface
• Aggregation of cells into microcolonies
• Production of polymeric matrix
• Formation of three-dimensional
structure known as biofilm

Development of
biofilm – timing

Development of
a biofilm

biology.binghamton.edu

Biofilm development, another
picture
webs.wichita.edu

Biofilm
development

www.ul.ie

Biofilm formation, another picture
www.uweb.engr.washington.edu

Importance of biofilm production in
bacteria
Bacteria may better regulate their quantity – in
the biofilm they inform each other by
production of various stuffs (quorum sensing)
Bacteria become more resistant to outer
influences:
– desinfectants
– antibiotics
– host immunity response
Biofilm is formed both by common flora bacteria (rather
positive for macrorganism and by pathogens

Biofilm

env.snu.ac.kr

Mechanisms influencing
bacterial resistance
•
•
•
•
•
•
•

Influence of surface charge
Decrease of growth rate
Penetration bariere
Non-homogenous matrix
Fenotypic differences
Intercelular signalisation
Immunity mechanizms...

www.dms-online.de

Biofilm

Biofilm eradication
• Antibiotic therapy often only supresses
symptomas of infection caused by cells
released from biofilm matrix and reacting with
immunity system. Cells fixed in biofilm matrix
cannot be destroyed by such therapy.
• To biofilm eradication we often to use high
ATB concentrations (monotherapy or
combinations), when treatment is not
effective, the biofilm focus should be
removed.
• In future we will possibly try to destroy the
biofilm, e. g. by enzymotherapy

Yeast Biofilm

www.ansci.wisc.edu

Prevention
• Catheters and bone cements
– made of new generation plastic material (risk
of adhesion and biofilm formation lower)
– with coloid silver and similar surface-active
compounds
– with antimicrobial substances, e. g.
• minocycline
• rifampicine

• Catheter washing
• Correct asepsis, decontamination methods etc.

Diagnostic and
experimental
methods for biofilm

Biofilm and microbiologic diagnostics
a) Biofilm assessment
aa) by phenotypic methods (Christensen‘s method,
Congo red agar cultivation)

Foto: Archiv Veroniky Holé

ab) by genotypic methods

b) Assesment of bacterial susceptibility in biofilm to
individual antibiotics or combinations (mostly MBEC)
c) Regarding to biofilm formation at common
bacteriological diagnostics, e. g. at venous catheter
cultivation we choose specific methods (see later)
instead of classic multiplication in broth

Microscopy of oral biofilm

Besides official methods for biofilm detection
there are also other methods how to visualise
biofilm.
For oral biofilm:
Gram stain may only visualise cell clusters (both G+
and G- ) and eventually macrooranism cells
(epitheliae etc.). Polysacharidic masses remain
invisible.
Alciane blue stain enables
visualisation of
polysaccharicic material, i. e.
the acellullar part of biofilm.
Cells are visualized by
negative staining.
Photo: Archive of Veronika Holá

Proof of influence of tooth cleaning
to oral biofilm
Photo: Archive of Veronika Holá



A volunteer has a
iodine solution or
pills with a stain
effecting to tooth
plaque.

Photo: Archive of Veronika Holá

The iodine is let to work
in oral cavity during
approx. 2 min.


Culture of biofilm producing
bacteria




In case of likelihood of biofilm formation, it
is usually necessary to perform special
methods for pre-processing the biological
material, that precede the proper culture
For central venous catheter culture, there exist
two methods. Both of them are better than
classical culture in broth without any preprocessing, sonification still remaining better
than the Maki method

Methods

• Classical broth culture: Bacteria in planctonic form

are released. Bacteria in form of a biofilm are
released. Bacteria in biofilm form are released less,
or not at all. As broth is used as multiplying medium,
we know nothing about its quantity (contamination ×
infection).

• Semiquantitative (Maki) method: It enables us
to assess catheter surface and semiquantitativelly
assess the finding, but we have no information about
intraluminal bacteria and bacteria are not necesarilly
released from the biofilm.
• Sonification: destroys biofilm on the catheter
surface and catheter lumen. Inoculation of a defined
specimen volume is a quantitative method, that
enables as to assess microbial amount.

Proof of influence of saccharides

presence to dental plaque formation
• The experiment has a simple principle.
One of oral bacteria is cultured on plastic
surface (simulating tooth surface) with
presence of various concetrations of
glucose and for various time value
• After the incubation, biofilm is visualised
using gentiane violet and its density
quantified as absorbance using a
spectrophotometre

To avoid accidental mistake, six adjacent
wells have always the same values of
both glc concentration and time

Old and new abbreviations in
antibiotic effect measuring
MIC – minimal inhibition concentration is
the growth limit of bacteria (the lowest
concentration that disables bacterial growth)
MBC – minimal bactericidal concentration
is the survival limit of bacteria (the lowest
concentration that kills bacteria). In viruses,
we would use „minimal virucidal“ etc.

MBIC – minimal biofilm inhibiting concentration
MBEC – minimal biofilm eradication
concentration

Diagnostic
methods MBEC
assessment

Kontrola růstu
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

MBEC … minimal
biofilm eradicating
concentration

(Another value exists:
MBIC … minimal
biofilm inhibitory
concentration – a value
not approved by all
scientists)

Photo: Archive of Veronika Holá

Photo: Archive of Veronika Holá

MIC versus MBEC
• While MIC determinates minimal
inhibitory concentration of atb in planctonic form,
MBEC shows us if eradication of bacterial biofilm is
present.
So it tells us more about effect of antibiotic on
normally living bacteria
• MBEC corresponts the lowest concentration of
antibiotic, where biofilm eradication is
proven (absence of living cell, no pH medium
change, the well remains red)

Differences in MIC, MBIC, MBEC
Porovnání MIC, MBIC a MBEC (log)
log
100000
10000
1000
100
10

MIC

MBIC

VAN

TEI

GEN

CIP

CLI

ERY

COT

TET

CMP

AMS

OXA

PEN

1

MBEC

Abbreviations: pen – penicilin, oxa – oxacilin, ams – ampicilin/sulbactam, cmp
- chloramphenicol, tet – tetracycline, cot – co-trimoxazole, ery – erythromycine,
cli – clindamycine, cip – ciprofloxacine, gen – gentamicine, tei – teicoplanine,

Diagnostic methods II.
• Values of MBEC are often over break point
for given antibiotics (bacterie are resistant to
them)
• Values of MBEC use to be several times
higher than MIC
• Microbes in biofilm are usually resistant even to
antibiotic combinations, the only possibility
is then biofilm focus removal (a catheter, joint
implants, tooth implants etc.)

The End
This slideshow was
prepared in cooperation of
ing. Veronika Holá, MUDr.
Lenka Černohorská, PhD.,
and MUDr. Ondřej
Zahradníček

(Student K. C.
several years ago
forgot to bring her
index, so she got
the credit in the
evening in a pub )
Photo: Archive of O. Z.